Neural contributions to concentric vs. eccentric exercise-induced strength loss

The purpose of this study was to examine the strength, electromyographic (EMG), and mechanomyographic (MMG) responses after workouts designed to elicit fatigue and muscle damage vs. only fatigue. Thirteen men (mean ± SD age = 23.7 ± 2.2 years) performed 6 sets of 10 maximal concentric isokinetic (CONexercise) or eccentric isokinetic (ECCexercise) muscle actions of the dominant forearm flexors on 2 separate days. Before (PRE) and after (POST) these workouts, peak torque (PT), surface EMG, and MMG signals were measured during maximal concentric isokinetic, eccentric isokinetic, and isometric muscle actions of the forearm flexors. The subjects also visited the laboratory for a control (CTL) visit with quiet resting between the PRE and POST measurements, rather than performing the CONexercise or ECCexercise. The results showed that there were significant 26 and 25% decreases in PT after the CONexercise and ECCexercise, respectively, and these decreases were statistically equivalent for the concentric, eccentric, and isometric muscle actions. There were also 19 and 23% reductions in normalized EMG amplitude after the CONexercise and ECCexercise, respectively, but no changes in EMG mean frequency (MNF), MMG amplitude, or MMG MNF. These findings demonstrated a neural component(s) to the strength decrement after CONexercise and ECCexercise. It is possible that after these 2 types of exercise, activation of free nerve endings that are sensitive to muscle damage and pH changes resulted in inhibition of alpha motor neurons, causing decreased muscle activation and torque. These findings suggest that training programs designed to minimize strength loss during competition should consider the fact that at least some of this loss is because of neural factors.     

Absolute quantification of somatic DNA alterations in human cancer

We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.     

Ebola Virus Genome Plasticity as a Marker of Its Passaging History: A Comparison of In Vitro Passaging to Non-Human Primate Infection

To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.     

Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology

The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes, including NANOG, SOX2, cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.     

WNT7B in fibroblastic foci of idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial pneumonia causing a loss of respiratory surface area due to a proliferative fibrotic response involving hyperplastic, hypertrophic, and metaplastic epithelium, cystic honeycomb change, septal expansion, and variable inflammation. Wnt (wingless) signaling glycoproteins are known to be involved in lung development and tissue repair, and are up-regulated in patients with IPF. Based on previous qRT-PCR data showing increased Wnt7B in lungs of IPF patients, a systematic, quantitative examination of its tissue site distribution was undertaken.

Methods

Tissue samples from the Lung Tissue Research Consortium (LTRC) of 39 patients diagnosed with mild to severe IPF/usual interstitial pneumonia (UIP) and 19 normal patients were examined for the immunolocalization of Wnt7B.

Results

In normal lung, moderate Wnt7B reactivity was confined to airway epithelium, smooth muscle of airways and vasculature, and macrophages. IPF lung showed strong Wnt7B reactivity in fibroblastic foci, dysplastic airway and alveolar epithelium, and in highly discrete subepithelial, basement membrane-associated regions. All reactive sites were sized and counted relative to specific microscopic regions. Those in the subepithelial sites were found in significantly greater numbers and larger relative area compared with the others. No reactive sites were present in normal patient controls.

Conclusions

The results demonstrate Wnt7B to be expressed at high concentrations in regions of active hyperplasia, metaplasia, and fibrotic change in IPF patients. In this context and its previously established biologic activities, Wnt7B would be expected to be of potential importance in the pathogenesis of IPF.     

Remote sensing to map influence of light pollution on Cory’s shearwater in São Miguel Island,Azores Archipelago

Global economic and population growth increase the extent and intensity of artificial night lighting. From an ecological perspective, this is light pollution, which causes changes in reproductive physiology, migration and foraging of many species and ultimately leads to loss of biodiversity. Some seabirds are intimately linked with the light features of their environments because they are nocturnally active. We report light-induced groundings of Cory’s shearwater (Calonectris diomedea) during a 2-year study (2008 and 2009) in São Miguel Island, in the Azores archipelago, and investigate the spatial correlation of locations of grounded birds with an annual composite of remotely sensed stable lights. Results indicate that 16.7% of fledglings are attracted to lights. The exposure of shearwater colonies in the study area to artificial night lighting is low overall. Four colonies account for 87% of the grounded birds. The distance each bird was found from the closest colony was best explained by the ratio of the satellite-measured light levels at the grounding spot to the light levels at the assigned colony of origin. These results demonstrate that satellite-observed nighttime lights are sufficient to assess risk to marine birds at the scale of oceanic islands and indicate their utility for monitoring the effectiveness of programs to manage lighting to reduce risk for these species and conducting global assessments of species vulnerability. To minimize the impact on Cory’s shearwater and other marine birds, we recommend measures such as reduction and control of lighting intensity near colony locations, while continuing and re-enforcing rescue campaigns.     

Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly(142) is critical for tetramerization of CTP synthase from Escherichia coli

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.     

Model‐guided design of ligand‐regulated RNAi for programmable control of gene expression

Progress in constructing biological networks will rely on the development of more advanced components that can be predictably modified to yield optimal system performance. We have engineered an RNA‐based platform, which we call an shRNA switch, that provides for integrated ligand control of RNA interference (RNAi) by modular coupling of an aptamer, competing strand, and small hairpin (sh)RNA stem into a single component that links ligand concentration and target gene expression levels. A combined experimental and mathematical modelling approach identified multiple tuning strategies and moves towards a predictable framework for the forward design of shRNA switches. The utility of our platform is highlighted by the demonstration of fine‐tuning, multi‐input control, and model‐guided design of shRNA switches with an optimized dynamic range. Thus, shRNA switches can serve as an advanced component for the construction of complex biological systems and offer a controlled means of activating RNAi in disease therapeutics.     

Thirteen polymorphic microsatellite DNA loci from whiptails of the genus Aspidoscelis (Teiidae: Squamata) and related cnemidophorine lizards

We describe polymerase chain reaction primers and amplification conditions for 13 microsatellite DNA loci isolated from two bisexual species of whiptail lizards Aspidoscelis costata huico and Aspidoscelis inornata. Primers were tested on either 16 or 48 individuals of A. c. huico and/or 26 individuals of A. inornata. Ten of the 13 primers were also tested against a panel of 31 additional whiptail taxa. We detected three to nine alleles per locus in A. c. huico and four to 19 alleles per locus in A. inornata, with observed heterozygosity ranging from 0.60 to 0.87 and from 0.15 to 1.00, respectively. These primers will be an important resource for surveys of genetic variation in these lizards.